Co-enzyme A&acetyl Co-enzyme A are very important biological compounds,& participate in amino acid, carbohydrate, and lipid metabolism. Research in these areas will be facilitated by rapid and accurate analysis of these compounds. Co-A has a reactive thiol group, to which acyl groups can be covalently linked to form thioesters during the acyl-group transfer reactions. High specific activity tritiated acetyl-CoA, therefore will be a valuable molecule for the specific labelling of enzymes and will allow receptor-binding studies to be carried out at lower concentrations. Tritiated Co-A is commercially available only at 2-10 Ci/mmole, but is very expensive. Our collaborators have synthesized it in their laboratory using tritiated acetic anhydride with a specific activity of 3-5 Ci/mmole. This level of radioactivity is not high enough to facilitate labelling and detection of lyzosomal membranes in their research program and a substrate with higher specific activity is required. This project was an excellent opportunity to examine the application of the newly developed N-tritioacetoxyphthalimide reagent for the labelling of thiol groups. In our exploratory experiments we applied the non-radioactive reagent to the S-acetylation of L-cycteine, using water as the reaction solvent. When one equivalent of N-acetoxyphthalimide was used as the acetylating reagent with one equivalent of L-cysteine as the substrate, N-acetyl-L-cysteine was the sole product, as shown by proton NMR and mass spectrometry. Reactiuon with two equivalents of the reagent generated N,S-diacetyl-L-cysteine under mildly basic conditions, as shown by proton NMR and mass spectrometry. Encouraged by these results, we reacted the reagent with the commercial Co-enzyme A sodium salt in water. This reaction gave a very clean product that was analyzed by proton NMR spectroscopy, and shown to be S-acetyl Co-A. A new HPLC method was developed for analysis of S-acetyl Co-A, and analysis of our acetylation product gave 80% chemical yield. Having established the reaction and analysis conditions, we are planning to prepare high specific activity tritiated Acetyl-Co-A in the near future. TEMLENGTH: